Korean Journal of Microbiology (2019) Vol. 55, No. 3, pp. 234-241 pISSN 0440-2413DOI https://doi.org/10.7845/kjm.2019.9056 eISSN 2383-9902Copyright ⓒ 2019, The Microbiological Society of Korea

Isolation and characterization of cellulolytic yeast belonging to

Moesziomyces sp. from the gut of Grasshopper§

Ju-Young Kim1, Hee-Young Jung

2,3, Jong-Seok Park

4, Sung-Jin Cho

4, Hoon Bok Lee

1, Gi-Ho Sung

5,6*,

Gayathri Subramani1* , and Myung Kyum Kim

1*

1Department of Bio & Environmental Technology, College of Natural Science, Seoul Women’s University, Seoul 01797, Republic

of Korea2College of Agricultural and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea

3Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Republic of Korea

4School of Biological Sciences, College of Natural Sciences, Chungbuk National University, Chungbuk, 28644, Republic of Korea5Institute for Healthcare and Life Science, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong

University, Incheon 22711, Republic of Korea6Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung 25601, Republic of Korea

메뚜기의 내장에서 분리한 Moesziomyces 속에 속하는 셀룰로오스 분해

효모의 분리 및 특성§

김주영1, 정희영2,3, 박종석4, 조성진4, 이훈복1, 성기호5,6*, 가야쓰리 수브라마니1* , 김명겸1*1서울여자대학교 자연과학대학,

2경북대학교 농업생명과학대학,

3경북대학교 식물의학연구소,

4충북대학교 자연과학대학 생물과학부,

5가톨릭관동대학교 국제성모병원 및 의과대학,

6강릉 가톨릭관동대학교 의과대학 미생물학교실

(Received June 10, 2019; Revised September 4, 2019; Accepted September 4, 2019)

*For correspondence. (G.H. Sung) E-mail: sung97330@gmail.com;

Tel.: +82-032-290-2772; Fax: +82-2-290-2614 /

(G. Subramani) E-mail: drgaya@swu.ac.kr;

Tel.: +82-2-970-5670; Fax: +82-2-970-5974 /

(M.K. Kim) E-mail: biotech@swu.ac.kr;

Tel.: +82-2-970-5667; Fax: +82-2-970-5974§Supplemental material for this article may be found at http://www.kjom.org.

An intensive interaction between yeasts and insects has

highlighted their relevance for attraction to food and for the

insect's development and behavior. Yeast associated in the gut

of insects secretes cellulase which aided in the food digestion

(cellulose degradation). Three strains of cellulose-degrading

yeast were isolated from the gut of adult grasshoppers collected

in Gyeonggi Province, South Korea. The strains ON22T, G10

T,

and G15T, showed positive cellulolytic activity in the car-

boxymethyl cellulose (CMC)-plate assay. The phylogenetic

tree based on sequence analysis of D1/D2 domains of the large

subunit rRNA gene and the internal transcribed spacer (ITS)

regions revealed that the strains ON22T (100 and 98.4%

sequence similarities in D1/D2 domains and ITS) and G10T

(99.8 and 99.5% in D1/D2 domain and ITS region) were most

closely related to the species Moesziomyces aphidis JCM

10318T; G15

T (100% in D1/D2 domains and ITS) belongs to

the species Moesziomyces antarcticus JCM 10317T, respec-

tively. Morphology and biochemical test results are provided in

the species description. Cellulase with its massive applicability

has been used in various industrial processes such as biofuels

like bioethanol productions. Therefore, this is the first report of

the cellulolytic yeast strains ON22T, G10

T, and G15

T related to

the genus Moesziomyces in the family Ustilaginaceae (Ustila-

ginales), in Korea.

Keywords: Moesziomyces sp., D1/D2 domain, grasshopper, ITS,

rRNA, yeast

Isolation and characterization of cellulolytic yeast strains ∙ 235

Korean Journal of Microbiology, Vol. 55, No. 3

Cellulose is the most abundant and renewable organic

material in the environment (Tomme et al., 1995). Cellulose

consists of glucose monomers, which is the abundantly

available source of sugar for fermentation and has a high

potential for bioconversion to important byproducts such as

ethanol. Inexpensive fuel can be supplied by selecting/using

highly potential cellulose-degrading microorganism in the

ethanol production. Cellulases are hydrolytic enzymes syn-

thesized by a large diversity of microorganisms including both

fungi and bacteria during their growth on cellulosic materials

(Lee et al., 2004). The best source of isolating these microbes

is from the gut of insects, such as, termites, bookworm and

cockroach thriving on cellulosic substances as their feed

(Dillon et al., 2004). Therefore, the endosymbiotic microbes

inside the insects are most capable of degrading cellulose. So

far, Chaetomium, Trichoderma, Fusarium, Myrothecium, Tri-

choderma. Penicillium and Aspergillus are some of the re-

ported fungal genera responsible for cellulosic biomass hydro-

lyzation (Romão-Dumaresq et al., 2016). Among them fungus

Trichoderma reesei is a well-known cellulolytic organism and

capable of producing a family of different cellulolytic enzy-

mes, including endoglucanases, exo-cellobiohydrolases, and β-

glucosidases (Ito et al., 2004). Endoglucanases can hydrolyze

amorphous, soluble, and substituted celluloses randomly

(Fujita et al., 2002).

The yeast species gained importance in the genetically

modified production of enzymes. For example, in Saccharo-

myces cerevisiae cellulase and β-glucosidase genes have been

over expressed to produce ethanol (Van Rensburg et al., 1998).

Also yeast strains exhibiting various proteins on the cell

surface have been developed by using genetic engineering

techniques (Takahashi et al., 2000). Microbial enzymes have

advantages over the animal and plant enzymes. They are

economical and can be produced on large scale within the

limited space and time. It can be easily extracted and purified.

They are capable of producing a wide variety of enzymes in a

wide range of environmental conditions, which shows genetic

flexibility (Trevan, 1987). Thus, our preliminary study on

isolating yeast species with cellulolytic activity will add

importance in the field of enzyme technology for the cellulase

production.

In this study, the yeast species ON22T, G10

T, and G15

T

belonging to the genus Moesziomyces, family Ustilaginaceae

are capable of degrading cellulose in CMC-plate assay. At

present this teleomorphic yeast contain five species M.

antarcticus, M. aphidis, M. bullatus, M. parantarcticus, and

M. rugulosus (Wang et al., 2015). Hence, it is the first report

of cellulase activity in this teleomorphic Moesziomyces species.

Materials and Methods

Yeast isolation and maintenance

The yeast strains were isolated from grasshoppers collected

from Onam-ri (37.6911° N, 127.2158° E), Gyeonggi Province,

South Korea. The grasshoppers bodies were frozen separately

at -20°C. During the experimental day of dissection, frozen

grasshoppers taken out from the freezer and instantly rinsed

with 70% ethanol. The exoskeleton of each grasshopper was

then cut along the side and the digestive system was extracted.

The whole guts were then stored in 1.5 ml microcentrifuge

tubes, the tissue was dissected. The samples were cut into

segments of 0.2~0.4 cm without surface-sterilization. Between

10 and 30 tissue segments were then evenly placed in 9 cm

diameter Petri dishes containing yeast extract-malt extract

(YM) agar plates (pH 3.7~5.0) (Difco) supplemented with

0.01% (w/v) chloramphenicol (Difco) and 0.02% (w/v) sodium

propionate (Difco), then incubated at 25 ºC in the dark. The

colonies appeared on the plates around a period of 3~5 days

were transferred to YM broth for growth. The strains were then

purified by repeated streaking of an isolated colony onto YM

agar followed by incubation at 25ºC. Purified yeast strains were

suspended in YM broth supplemented with 10% glycerol (v/v)

and maintained at -80ºC.

Phenotypic characterization

Morphology, physiology, and biochemical studies were

performed following the methods described by Kurtzman et al.

(2011). Formation of pseudohyphae and true hyphae was

investigated by cultivation on potato dextrose agar (PDA,

Difco) in the slide culture at 25ºC for 2 weeks. The results were

observed in light microscopy. Growth at various temperatures

(15, 20, 25, 30, 32, 40, and 42°C) and NaCl concentrations was

236 ∙ Kim et al.

미생물학회지 제55권 제3호

(A) (B) (C)

Fig. 1. Morphology of the cell on YM agar after 3 days at 20°C. (A) ON22T (B) G10

T and (C) G15

T. Scale bar, 10 µm.

determined by cultivating yeast in YM agar.

Cellulase activity in CMC plate assay

The cellulolytic degrading yeast strains were screened using

CMC (Carboxymethyl cellulose) plate-based clearing assay as

described by Johnsen and Krause (2014) with some modified

with an antibiotic identification as given below. The Czapek-

Dox-CMC medium containing KH2PO4 (1 g/L), MgSO4 ․ 7H20

(0.5 g/L), KCl (0.5 g/L), FeSO4 ․ 7H2O (0.01 g/L), CMC (30

g/L), NaNO3 (2 g/L), noble agar (20 g/L), and antibiotics (chlo-

ramphenicol) (0.02%) were poured into Petri dishes and was

allowed to polymerize at room temperature overnight. The

yeast strains were streaked onto the plates and were incubated

at 27°C for 12~16 h and then hydrolysis zones were visualized

by flooding of the plates/wells with Gram’s iodine (2 g pota-

ssium iodide and 1 g iodine in 300 ml water) for 5 min followed

by a rinse with deionized water. CMC-free plates were (non-

substrate) were used as controls in all experiments. All the

chemicals were purchased from Difco.

DNA sequencing and phylogenetic analysis

DNA was extracted from yeast and purified using the CTAB

method (Cubero et al., 1998). The D1/D2 domain was ampli-

fied with the primers NL1 and NL4 as described by Kurtzman

and Robnett (1998). Amplification of the D1/D2 domain was

performed as follows: 94ºC for 6 min, followed by 40 cycles of

94ºC for 60 sec, 50ºC for 60 sec, and 72ºC for 60 sec; and a final

extension at 72ºC for 5 min. The ITS (ITS1–5.8 S–ITS2) region

of the rRNA gene was amplified with the primers ITS1F and

ITS4 as described by White et al. (1990). Amplification of the

ITS region was performed using the following conditions:

95ºC for 3 min, followed by 37 cycles of 94ºC for 30 sec, 52ºC

for 30 sec, and 72ºC for 30 sec; and a final extension at 72ºC for

10 min. The related D1/D2 domain and ITS region sequences

were obtained from GenBank database and edited with the

BioEdit program (Hall et al., 1999). Multiple alignments were

performed with the CLUSTAL X program (Thompson et al.,

1997) and the alignment was manually verified prior to the

construction of phylogenetic trees. Phylogenetic trees were

constructed using the MEGA 7 program (Kumar et al., 2016)

by the neighbour-joining method (Saitou and Nei, 1987) and

maximum likelihood (Fitch, 1971). During the phylogenetic

analysis, evolutionary distances were calculated using the

Kimura two-parameter model (Kimura, 1980), and bootstrap

values were calculated based on 1,000 replications (Felsenstein,

1985).

Results and Discussion

Morphology and physiology of yeast strains

The yeast strains ON22T, G10

T, and G15

T are selected based

on their cellulase activity. The cells of the strains ON22T and

G10T were cylindrical or fusiform with 2.0–3.5 × 5.4–9.8 µm

and 3.2–4.0 × 6.0–10.2 µm in size and are occurred either in

single or pairs (Fig. 1A–C) after grown in YM broth at 20°C for

5 days. On YM agar, after 5 days the colonies are light bro-

wnish, butyrous and wrinkled. The margin is eroded. Budding

is polar on short stalks. Growth was observed at 20, 25, 30, and

37°C. Growth was observed in 50% glucose. Cells are positive

to diazonium blue B. Production of starch-like substances are

absent. The type strain of Moesziomyces aphidis JCM 10318

showed similar pattern of results reported by Parahym et al.

Isolation and characterization of cellulolytic yeast strains ∙ 237

Korean Journal of Microbiology, Vol. 55, No. 3

(2013).

Whereas, in YM broth at 20°C for 5 days. the strain G15T

showed cylindrical or fusiform cells with 1.3~2.0 × 4.5~5.6 µm

in size and are occurred in singles. They showed polar budding.

On YM agar, at 20°C after 5 days the colonies are cream color,

smooth and glistening with fringed margin. Growth was

observed at 20, 25, 30, and 37°C. Growth at 50% glucose was

positive. Cells are negative to diazonium blue B. Starch-like

substances are not produced. The type strain of Moesziomyces

antarcticus JCM 10317T also reported with similar pattern of

results (Wei et al., 2005). Fermentation is negative for all the

three strains. Pseudohyphae was absent. Based on the

morphology and physiology characteristic the strains isolated

in Korea are belonging to the species of Moesziomyces.

Cellulase activity in CMC plate assay

The strains ON22T, G10

T, and G15

T showed clear zone area

on the surface of the medium after staining with iodine solution

(Supplementary data Fig. S1), which proved the degradation

efficiency of the strains. The strain G1 is used as a negative

control, which was a non-cellulolytic strain. The results were

compared by using CMC free medium as well without yeast

cells. These plates assay results provided advantage of ob-

serving the results visually using smaller sample volumes. The

cellulolytic activity can be quantified by a variety of methods

that have been summarized in papers (Zhang et al., 2009). The

previous methods used crystalline cellulose, but the degra-

dation rates were very slow, then most assays were adapted to

use more easily degradable soluble cellulose derivatives like

carboxymethylcellulose (CMC) (Yeoh et al., 1985). Therefore,

preliminary screening for extracellular cellulase production by

bacteria and fungi is often done on agar plates containing CMC

as substrate (Dashtban et al., 2010). Following the results, the

screening plate assay of the strains ON22T, G10

T, and G15

T are

selected as a candidate producer of cellulase, which showed

hydrolytic activity on carboxymethyl cellulose (CMC).

Sequence analysis

The three strains ON22T, G10

T, and G15

T recognized from

morphological characterization were confirmed by the sequence

analysis of D1/D2 domains and ITS regions. The sequence

analysis revealed the assignment of the strains ON22T, G10

T,

and G15T to the genus Moesziomyces. Therefore, strain ON22

T

shared 100% and 98.4% sequence similarity (0% and 0.5%

substitutions) in D1/D2 domain and ITS region with Moes-

ziomyces aphidis JCM 10318T; as well strain G10

T showed

99.8% and 99.5% (0% and 0.2% substitutions) in D1/D2

domain and ITS region with Moesziomyces aphidis JCM

10318T. Strain G15

T shared 100% sequence similarities in both

the D1/D2 domains and ITS with Moesziomyces antarcticus

JCM 10317T, respectively. Further the analysis proved the

theories of Boekhout and Fell (1998) and Sugita et al. (1999)

therefore, conspecific strains have a less than 1% nucleotide

difference in their ITS regions. Recently, the taxonomic

thresholds predicted to discriminate yeast species were 98.4%

for ITS and 99.5% for D1/D2 domain of the LSU (Vu et al.,

2016). The type strain of M. antarcticus previously described

as Pseudozyma antarctica was isolated from lake sediment in

Antarctica and was initially classified in the genus Sporo-

bolomyces. However, morphologically it differed from other

Sporobolomyces species by lacking ballistospores (Goto et al.,

1969). More recently, the genus Pseudozyma is a polyphyletic

anamorphic genus reclassified with species occurring in

clusters together with teleomorphic species of Moesziomyces.

(Wang et al., 2015).

The phylogenetic analysis using neighbor joining method

on the combined sequence of D1/D2 domain of LSU rDNA and

ITS regions showed the strain ON22T and G10

T formed in-

dependent clade with Moesziomyces aphidis JCM 10318T and

G15T

formed independent clade with Moesziomyces antar-

cticus JCM 10317T (Fig. 2). Thus, the phylogenetic trees added

additional evidence for the strain’s taxonomic positions.

Comparison of biochemical analysis between strains

ON22T, G10

T, and G15

T

The strains ON22T, G10

T, and G15

T lack fermentation of

carbon sources, which is the major property of this genus

previously reported by Wei et al. (2005) and Wang et al.

(2006). The major similarity in assimilation tests in between

the species were given in the Table 1. The strain G15T differed

from ON22T and G10

T by showing negative to Diazonium blue

B. The strain G10T differed from ON22

T by assimilating inulin,

cellobiose, L-sorbose, D-ribose, xylitol and DL-lactate. More-

over, ON22T and G10

T displays similar physiological charac-

238 ∙ Kim et al.

미생물학회지 제55권 제3호

Table 1. Difference in physiological characteristics between the strains

ON22T, G10

T, and G15

T. All the data obtained in this study. Growth

reactions: -, no growth; w, weak growth; +, strong growth.

Characteristic 1 2 3

Colony color Light brown Light brown Cream

Diazonium blue B reaction + + -

Assimilation

α-Methyl-D-glucoside + + -

Salicin w w -

D-Gluconate + + w

D-Ribose + w -

Inulin - + -

Fig. 2. Phylogenetic tree using the neighbour-joining and maximum likelihood methods based on the sequences of the ITS regions and LSU D1/D2 domain

of strains ON22T, G10

T, and G15

T with the members of closely related taxas. Bootstrap values based on 1,000 replications are shown at the branch nodes.

Farysizyma itapuensis CBS 10428T (DQ767831) is used as an outgroup. Bar, 0.02 substitutions per nucleotide position.

teristics in the assimilation with the identified species M.

alphidis (Table 2). The strain G15T assimilation results with M.

antarticus were compared in Table 3. These strains have Q10

as their major quinone. Therefore, assimilation tests and other

growth characteristics are important for the identification of

Moesziomyces species.

Conclusion

Most of the insects that depend exclusively on nutritional

restricted diets such as plant sap, and woody material, commonly

possess obligate mutualistic endosymbionts (gut microbes)

involved in the provision of essential nutrients or in the

degradation of food materials. While studying and selecting

yeasts for a specific function and classification, identification

of the isolates is important. Isolation of cellulolytic yeast

strains gained importance for their incredible usage in biofuel

and bioethanol productions. Based on the theories on sequence

analysis [Boekhout and Fell (1998), Sugita et al. (1999), and

Isolation and characterization of cellulolytic yeast strains ∙ 239

Korean Journal of Microbiology, Vol. 55, No. 3

Table 2. Physiological and biochemical tests capable of showing similarity

between strains of ON22T, G10

T and Moesziomyces aphidis

Characteristic 1 2 3

Glucose (50%) + + +

Production of starch-like substance - - -

Assimilation

Cellobiose - + -

Galactose + + +

Glucose + + +

Inulin - + -

Lactose + + +

Maltose + + +

Melezitose + + +

Melibiose + + +

α-Methyl-D-glucoside + + +

Raffinose + +

L-Rhamnose + + +

D-Ribose + w -

Salicin W w +

Soluble Starch - - +

L-Sorbose W + +

Trehalose + + +

Citrate - w -

D-Mannitol + + +

D-Gluconate + + w

Glucanolactone + + +

D-Glucosamine + + +

D,L-Lactate + w w

Galactitol - w -

Erythritol + + +

Ethanol + +

D-Glucitol + + +

Cadaverine + + +

Potassium Nitrate + + +

Sodium nitrate - - -

Species: 1, ON22T; 2, G10

T; 3, Moesziomyces aphidis

Growth reactions: -, no growth; w, weak growth; +, strong growth.

Moesziomyces aphidis - data obtained from Wang et al. (2006).

Table 3. Physiological and biochemical tests capable of showing

similarity between strains of G15T and Moesziomyces antarticus

Characteristic 1 2

Glucose (50%) + +

Production of starch-like substance - -

Assimilation

Cellobiose W +

Galactose + +

L-Arabinose + +

D-Arabinose + w

Glucose + +

Inulin - -

Lactose + +

Maltose + +

Melezitose + +

Melibiose + +

α-Methyl-D-glucoside - +

L-Rhamnose + +

D-Ribose - +

Salicin - w

Soluble Starch - +

L-Sorbose + +

Sucrose + +

Trehalose + +

Citrate - w

D-Gluconate W s

Glucanolactone + +

D-Glucosamine + +

myo-inositol W +

Galactitol - -

Erythritol + s

Methanol - -

Ethanol + +

D-Glucitol + +

L-Lysine + ND

Mannitol + +

Ribitol - w

Cadaverine + +

Potassium Nitrate + ND

Sodium nitrate - ND

Species 1, G10T; 2, Moesziomyces antarticus

Growth reactions: -, no growth; w, weak growth; +, strong growth; ND, not

determined; s, slow positive.

Moesziomyces antarticus - data obtained from Wei et al. (2005).

Vu et al. (2016)] and polyphasic taxonomic approaches, the

strains ON22T and G10

T was identified as Moesziomyces aphidis

and the strain G15T

as Moesziomyces antarcticus.

Nucleotide sequence accession number and culture

deposition

The accession numbers of D1/D2 domain of ON22T, G10

T,

and G15T were MK205287, MK203814, and MK212920. The

accession number of ITS regions of ON22T, G10

T, and G15

T

240 ∙ Kim et al.

미생물학회지 제55권 제3호

were MK212919, MN038047 and MK212921.

The yeast strains ON22T, G10

T, and G15

T were deposited in

Korean Collection for Type Cultures, Korea. The KCTC num-

bers for the strains were ON22T (= KCTC 27804

T), G10

T (=

KCTC 2780T), and G15

T (= KCTC 27802

T).

적 요

효모와 곤충 간의 집중적인 상호 작용은 곤충의 먹이에 대한

유인과 발달 및 행동에 대한 관련성을 보였다. 곤충 내장에서

분리된 효모는 먹이의 소화를 돕는 셀룰라아제(셀룰로오스 분

해)를 분비한다. 한국의 경기도에서 수집한 메뚜기의 장에서

셀룰로오스를 분해하는 효모 세 균주를 분리 하였다. 효모 균

주의 cellulase 활성을 확인하기 위해, 카르복시 메틸 셀룰로즈

(CMC)를 함유하는 배지로 플레이트상의 투명한 영역을 요오

드 용액을 사용하여 관찰하였다. 효모 ON22T, G10

T 및 G15

T균

주는 CMC-플레이트 분석에서 양성 셀룰로오스 활성을 나타

냈다. Large subunit rDNA 유전자와 Internal transcribed spacer

(ITS) 영역의 D1/D2 영역의 서열 분석에 기초한 계통수를 통

해 ON22T와 G10

T 균주가 Moesziomyces aphidis JCM 10318

(D1/D2 영역에서 각 100%와 99.8%, ITS 영역에서 각 98.4%

및 99.5% 서열유사성)와 가장 가깝고 G15는 Moesziomyces

antarcticus JCM 10317T 종 (D1/D2 영역에서 100%, ITS에서

100% 서열 유사성)에 속한다는 것을 밝혔다. 셀룰라아제는 바

이오 에탄올 생산과 같은 바이오 연료와 같은 다양한 산업 공정

에서 사용되고 있다. 따라서, 셀룰로오스 분해 미생물의 분리 및

연구는 중요성을 갖게 되었다. 이 논문은 한국의 Moesziomyces

속의 셀룰로오스 분해 효모 균주인 ON22T, G10

T, G15

T에 대

한 첫 번째 보고이다.

Author’s contributions

All authors equally contributed in this work.

Conflict of Interest

All authors certify that there is no conflict of interest with

any financial organization regarding the material discussed in

the manuscript.

Acknowledgements

This work was supported by a research grant from Seoul

Women’s University (2019) and a grant from the National

Institute of Biological Resources (NIBR), funded by the

Ministry of Environment (MOE) of the Republic of Korea

(NIBR201839201, 2018 Graduate Program of Undiscovered

Taxa).

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